Players reported limited ROM in ADF-KF, HE, HAB-HF, HF-KE, and KF; and asymmetric ROM mainly in HE, ADF-KE, KF, ADF-KF, and HF-KE. In conclusion, this study provides gender-specific lower extremity flexibility profile scores in BPs that can help athletic trainers and athletic and conditioning trainers to identify those players who are at higher risk of injury due to abnormal ROM scores.Analysis of the flexibility profile of basketball players (BPs) can reveal differences in range of motion (ROM) by gender and also identify those players who are at higher risk for sports injuries. A descriptive observational study was conducted to determine the lower extremity flexibility profile of sixty-four basketball players and gender differences to identify players at higher risk of injury due to limited and asymmetric ROM in one or more movements. Participants: Sixty-four  competitive athletes from the national leagues of the Spanish basketball league system participated in the present study (power of sample size ≥0.99). The eight passive ROM tests of the hip, knee and ankle were assessed using the ROM-SPORT battery. Each player completed a questionnaire on age, basic anthropometric data, dominant extremities, and training and sport-related variables. The lower extremity flexibility profile was established at 15° and 10° hip extension (HE), 39° and 38° ankle dorsiflexion with knee extended (ADF-KE), 40° and 39° ankle dorsiflexion with knee flexed (ADF-KF), 43° and 43° hip abduction (HAB), 75° and 61° hip abduction with the hip flexed (HAB-HF), 78° and 83° hip flexion with the knee extended (HF-KE), 134° and 120° knee flexion (KF), and 145° and 144° hip flexion (HF) by male and female basketball players, respectively. Sex differences in HE, HAB-HF, and KF were observed in BPs ( Muscle extensibility is defined as the ability of some muscle tissue components to deform by increasing their length under the influence of an external and internal torque ,2. ChangThe method of measuring ROM has been established internationally by institutions such as the American Academy of Orthopaedic Surgeons  and the Knowing the reference values that define the lower extremity flexibility profile for their sport is of great interest to athletic trainers and athletic and conditioning trainers ,24. The At the same time, limited ROM due to muscle tightness ,28,34,35To our knowledge, previous studies have not identified lower extremity-based specificity analysis ROM for basketball players (BPs). Accordingly, the aim of the current study was twofold: (1) to define the lower extremity flexibility profile of sixty-four BPs along with gender differences, and (2) to identify players at higher risk of injury due to limited ROM or inter-limb ROM asymmetry in one or more movements.This investigation (ID: 1702/2017) was approved by the Institutional Ethics Committee of the College of Murcia (Spain) and complied with the World Medical Association Code of Ethics, Declaration of Helsinki.Seventy-three BPs from four basketball teams were initially recruited for this descriptive observational study. The players played in the second  and fourth  levels of the Spanish basketball league. In order to recruit the BPs to participate in our study, the researchers contacted the coaches one month before the assessment session. One week prior to the evaluation, an investigator met with the technical staff and BPs to explain the objectives of the study. This was also used to familiarize the BPs with the ROM tests. Familiarization with the testing procedures took place during the BPs’ first visit to the sports center during peak competition time. During the second visit, each player signed an informed consent form and completed the questionnaire that included demographic, anthropometric, and basketball training data. In addition, during the competition period, an assessment of essential lower limb movements was performed using the eight ROM tests from the ROM-SPORT battery .http://www.randomizer.org to avoid bias that might occur if the measurements were taken in a particular order. The average of the two measurements was used for further statistical analysis. A third measurement was taken if the investigators found a variation of more than 5% between these two measurements of any test to identify the error measurement. There was a pause of more than one minute between repetitions and two minutes between each test. The data were then analyzed to confirm or reject the null hypothesis.Players were instructed not to engage in vigorous physical exertion 48 h prior to the study and were informed that they could withdraw from the study at any time. BPs performed a dynamic warm-up of approximately 20 min, following recommendations from a previous study . All ROMLater, the lower extremity flexibility profile was determined and gender differences between BPs were analyzed. To complete the comprehensive analysis, each player’s ROM values were compared to reference values  to identify players who were ROM limited by muscle tightness and inter-limb ROM asymmetry.Sixty-four BPs aged between 17 and 31 years (average of 22.6 ± 2.9 years) participated as volunteers in this study and had an average of 12 years (12.4 ± 3.9 years) of basketball experience . ThirteeThe power of the sample size for this study was analyzed as described in the Statistical Analysis section.Two exercise and sports science graduates with at least 15 years of experience in musculoskeletal assessment conducted the data collection for this study. They were the same examiners and had the same competencies in assessing ROM. The principal examiner performed the ROM tests while the assistant examiner reviewed the compensatory movements and recorded the data . In a doThe survey included questions about self-reported age, basic anthropometric data , dominant laterality (defined as the leg you would use for one-legged jumps to achieve maximum jump height), training, and sport-related variables . The data in the surveys were cross-checked with the basketball coach to increase objectivity. If the coach and player data did not match, club or federation records were used. The examiners evaluated the anthropometric measurements and verified the information provided in the questionnaires.The maximum passive hip, one knee and ankle ROMs were assessed using the ROM-SPORT battery . This baFirst, the assumptions about the normality of the data were tested using the Shapiro–Wilk normality test. Descriptive statistics with mean and standard deviation were calculated for all variables tested.t-test for independent samples was conducted to determine possible gender differences in the demographic and athletic variables. A post hoc sample size calculation was performed using the software package G*Power 3.1.9.7.  using a sample size of 33 female and 31 male basketball players, an alpha level of p < 0.05, effect size (Hedges’ g), and Student’s t-test for independent samples  [Second, a Student’s igure 2) .t-test was calculated to estimate inter-limb ROM asymmetry. To assess the effect size of the difference, Hedges’ g was calculated from the values of the paired samples t-tests and the effect was classified as no effect (g < 0.2), small: (g = 0.2 to 0.59), moderate (g = 0.6 to 1.19), large (g = 1.20 to 2.00), very large (g = 2.00 to 3.99), extremely large (g > 4.00) [Paired samples  > 4.00) . The low > 4.00) .t-test was performed to assess differences between the ROM of younger and older players.A k-means cluster analysis was performed to classify participants between younger and older players. An independent-samples t-test was performed to assess differences between normal and limited ROM groups; the Hedges g effect size was also considered. Statistical analyses were performed using SPSS software , with a significance level of at least five percent.Cut-off values for risk factors according to previous prospective cohort studies were used to identify athletes at higher risk of injury with normal vs. inter-limb ROM asymmetry  and 10° for the high ROM values as HF-KE, KF, and HF-KF) ,43,44,45The initial sample consisted of 73 players. Nine players were withdrawn from the study by the researchers. The reasons were that the players had not completed the questionnaire correctly, had not signed the informed consent form, or had received long-term rehabilitation treatment (ankle sprain and low back pain) in the past six months, including flexibility training.The variables  identified in this study resulted in a sample size significance of 0.99 for HE ROM, 1.00 for HAB-HF ROM and 0.99 for KF ROM.p = 0.029); however, the effect size of the effect found between both sides of the body was small (Hedges’ g = −0.34). Inter-limb ROM asymmetry was found only for the quadriceps in KF ROM . p = 0.01; Hedges’ g = 1.04)), HAB-HF ROM ) and KF ROM ) with moderate, very large and large effect sizes, respectively. Male players showed higher values than female players in all three ROMs.Differences were found between the group of young players (<23 years) and the group of older players (≤23 years) in the KF ROM  of the players in HE (iliopsoas), 24 (37.5%) of the players in ADF-KF (soleus), 44 (68.8%) of the players in HAB-HF (monoarticular adductors), 39 (60.9%) of the players in HF-KE (hamstrings), 37 (57.8%) of the players for KF (quadriceps), and 18 (28.1%) of the players for HF ROM (gluteus maximus) showed limited ROM .Individual analysis of inter-limb ROM asymmetry revealed a slightly greater number of lower values in the dominant limb compared to the same movement in the contralateral limb . Lower vThe present study is the first to describe the lower extremity flexibility profile in 64 BPs of both sexes using a standardised procedure. In general, the results suggest that the ROM-SPORT battery determines the first reference values of BPs competing in the national-tier leagues of the Spanish basketball league system. However, differences were observed between younger and older players in KF ROM, which is consistent with the results of previous studies ,16. The Most authors who have studied the flexibility of BPs have not addressed the analysis of sex differences ,55,56,57Second, our study demonstrates a comprehensive analysis of flexibility that provides athletic trainers and athletic and conditioning trainers a simple, individualised strategy to improve athletic performance and minimise athletic injuries in players. This comprehensive analysis observed limited ROM with a frequency of 28.1% to 68.8% of BPs for ADF-KF ROM (soleus), KF ROM (quadriceps), HF-KE ROM (hamstrings), HAB-HF ROM (monoarticular adductors), and HE ROM (iliopsoas). The cut-off values for this study are based on reference values from previous studies or clinical experience. In this sense, Backman and Danielson  presente95) of the ROM-SPORT tests is between 4° to 7° [Regarding the inter-limb ROM asymmetry, the results on the magnitude of the standardized mean differences show no inter-limb ROM differences, except for the KF ROM (quadriceps). The mean distance inter-limb ROM in the studied movements was 3° , 4° , and 5° (HF). These results are common in most studies calculating sports inter-limb ROM asymmetry. This result should not be considered valid because the sensitivity of the inclinometer is 2°, and the maximum detectable change , ADF-KE ROM (gastrocnemius), KF ROM (quadriceps), ADF-KF ROM (soleus), and HF-KE ROM (hamstrings). Compared to other team sports, the results for the frequency of inter-limb ROM asymmetry are lower than in soccer ,31, futsFuture research should focus on determining reference values in basketball with a representative sample of players from teams playing in a league. Prospective cohort studies are needed to determine the cut-off value for limited ROM and inter-limb ROM asymmetry for each test to identify BPs most likely to develop pain or injury. In addition, the reference values should be validated with a multifactorial assessment that includes physical and sports performance tests. In this regard, we recommend that researchers focus on the development and evaluation of pre-participation screening and injury prevention programs through high-quality randomised controlled trials targeting athletes at higher risk for basketball injury.Athletic trainers and athletic and conditioning trainers have a valuable tool in the ROM-SPORT battery to monitoring the ROM and conditioning in BPs. The normative data presented in this study serve as a baseline for evaluating ROM performance in BPs. A stretching routine could be developed for those players whose values fall below the basketball reference values. The design of this routine should also consider gender differences for athletes in HE, HAB-HF and KF ROMs. A comprehensive analysis may also help athletic trainers and athletic and conditioning trainers to identify musculoskeletal adaptations for sport, e.g., limited ROM by muscle tightness and inter-limb ROM asymmetry in BPs. This individualised ROM analysis is a useful strategy to improve athletic performance and reduce injury risk in competitive basketball players. However, this strategy will always be most effective when a multicomponent training program combining flexibility, strength, and neuromotor is used in basketball players.The lower extremity flexibility profiles of the sixty-four basketball players were 15° and 10° HE, 39° and 38° ADF-KE, 40° and 39° ADF-KF, 43° and 43° HAB, 75° and 61° HAB-HF, 78° and 83° HF-KE, 134° and 120° KF, and 145° and 144° HF ROMs of the male and female basketball players, respectively. Comparison of the two lower extremity flexibility profiles shows gender differences for the HE, HAB-HF, and KF ROMs. Basketball players mainly reported muscle tightness in ADF-KF, KF, HAB -HF, HF-KE, HF, and HE ROMs, and inter-limb ROM asymmetry mainly in HE, ADF-KE, KF, ADF-KF, and HF-KE ROMs. This study provides gender-specific lower extremity flexibility profile values in BPs that can help athletic trainers and athletic and conditioning trainers to identify those players who are at higher risk of injury due to abnormal ROM values. Reduction in injury risk can be achieved when professionals use a multicomponent training program that includes flexibility, strength, and neuromotor.


Background: An acute respiratory distress syndrome (ARDS) is caused by the increased amounts of pro-inflammatory cytokines and neutrophil-mediated tissue injury. To date, there is no effective treatment for the ARDS available, while the need for one is growing due to the most severe complications of the current coronavirus disease-2019 (COVID-19) pandemic. The human astrocytes (AstroRx) have shown immunomodulatory properties in the central nervous system (CNS). This study aimed to evaluate the capacity of astrocytes to decrease lung inflammation and to be applied as a treatment therapy in ARDS.Methods: First, we assessed the ability of clinical-grade AstroRx to suppress T-cell proliferation in a mixed lymphocyte reaction test. Next, we tested the therapeutical potential of AstroRx cells in a lipopolysaccharide (LPS)-based ARDS mouse model by injecting AstroRx intravenously (i.v). We determined the degree of lung injury by using a severity scoring scale of 0–2, based on the American Thoracic Society. The scoring measured the presence of neutrophils, fibrin deposits, and the thickening of alveolar walls. The state of inflammation was further assessed by quantifying the immune-cell infiltration to the bronchoalveolar lavage fluid (BALF) and by the presence of proinflammatory cytokines and chemokines in the BALF and serum.Results: We detected that AstroRx cells were capable to suppress T-cell proliferation in vitro after exposure to the mitogen concanavalin A (ConA). In vivo, AstroRx cells were able to lower the degree of lung injury in LPS-treated animals compared with the sham injected animals (P = 0.039). In this study, 30% of AstroRx treated mice showed no lung lesions (responder mice), these mice presented a steady number of eosinophils, T cells, and neutrophils comparable with the level of naïve control mice. The inflammatory cytokines and chemokines, such as TNFα, IL1b, IL-6, and CXCL1, were also kept in check in responder AstroRx-treated mice and were not upregulated as in the sham-injected mice (P < 0.05). As a result, the LPS-treated ARDS mice had a higher survival rate when they were treated with AstroRx.Conclusions: Our results demonstrate that the immunosuppressive activity of AstroRx cells support the application of AstroRx cells as a cell therapy treatment for ARDS. The immunoregulatory activity may also be a part of the mechanism of action of AstroRx reported in the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. An acute respiratory distress syndrome (ARDS) is a form of progressive hypoxemia respiratory failure and pulmonary edema in the absence of heart failure . The etiCurrent management for COVID-19  infected patients with severe pneumonia and ARDS remains supportive, such as the use of anti-infection drugs, intubated ventilator-assisted breathing therapy, and extracorporeal membrane oxygenation (ECMO) –16. SincAnother approach using astrocytes is presented as they are the most abundant glial cell in the central nervous system (CNS). Astrocytes regulate the concentration of different neurotransmitters and ions, supply various metabolites and energy, regulate osmolarity, modulate synaptic activity, secrete neurotrophic and neuroprotective factors, promote neurogenesis –23, and in vitro by evaluating T-cell proliferation in a mixed lymphocyte reaction test. Then, we assessed the effect of intravenous (i.v.) AstroRx injection on lung injury, lung inflammation, and survival in a lipopolysaccharide (LPS)-based ARDS mouse model. Altogether our results indicate that AstroRx has the potential to maintain immune homeostasis in the lung and thereby increase the chance of survival.In this current study, the effect of astrocytes on ARDS is investigated for the first time. We deciphered the immunomodulatory effect of AstroRx first in vitro and in the ALS animal models -derived astrocytes (AstroRx) protect MNs l models . In thesl models  and did l models . The Ast+/CD25+), which decreased by about 15% in the LNC:AstroRx® (at a ratio of 10:1) co-culture. A greater inhibitory effect was achieved when using a ratio of 5:1 of LNC:AstroRx culture, which decreased T-cell population by 42%. This effect was not observed when only AstroRx-conditioned medium was added . Without ConA, the percentage of human proliferating cells among the T cells was below 1%, the addition of different amounts of AstroRx cells to LNCs in the absence of ConA did not affect the percentage of proliferating T cells (not shown). In the presence of ConA, 40% of T cells were BrdU+ and proliferating. As a positive inhibitory control, we used mouse neural precursor cells (mNPCs), which were previously described for reducing T-cell proliferation . Naïve (n = 4), untreated mice and sham-injected LPS-treated mice (n = 10) served as the negative and positive controls, respectively. The treatment with AstroRx at both cell concentrations increased the survival of LPS-induced mice compared with the sham-injected animals . According to lung score, the animals within one group were categorized into the responders (lung score ≤2) and non-responders (lung score >2). The percentage of responders was higher among the AstroRx-treated mice compared with the sham injected mice .p = 0.0274 for neutrophils and macrophages and p = 0.0206 for T cells). The effect of AstroRx on the immune cells became even clearer when we analyzed the immune profile differentially in the responder and non-responder mice (P = 0.0286). The responder mice (lung score ≤2) among the AstroRx-treated LPS-injected mice had an immune profile that was similar to one of the naïve non-LPS-injected mice in terms of cell proportion and a general low-level of absolute cell number. While the non-responder mice displayed an immune profile that resembled the sham-injected mice with a general heavy infiltration of immune cells and a specifically massive invasion of neutrophils.In the sham-injected ARDS mice, most of the cells in the BALF consisted of neutrophils (>92%), while the immune profile of high-dose of AstroRx shift toward naïve mice the immune profile (P = 0.13 and higher). But when we dissected the group again into responders and non-responders, it became clear, that the responders among the AstroRx treated mice showed low expression of TNFa, IL1b, IL-6, IL-5, CC2, and CXCL1 ; 500k responders vs. non-responders: P = 0.0091 (for TNFa), P = 0.0286 for all other cytokines). Strikingly, their levels were similar to the levels in naïve, healthy mice. At the same time, the non-responder mice displayed cytokine/chemokine levels in the range of sham-injected mice. The same phenomenon was observed in blood serum as well (P = 0.0121 (for TNFa and CXCL1); 500k responders vs. non-responders P = 0.0235 [for TNFa and CXCL1)]. Altogether, our data showed that AstroRx had the capability to reduce the infiltration of immune cells to the lungs and to prevent the rising of a cytokine storm in the responder mice. It is reasonable to assume that this capability contributed to the better survival rate among the AstroRx-treated mice.As the immune profile is not only determined by the cells but also by molecules, we next analyzed the presence of cytokines and chemokines in the BALF and serum. At first sight, the expression of inflammatory cytokines and chemokines was quite heterogeneous within the group of AstroRx-treated animals  B27 supplement, 1% glutamax, and 1.5% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.4 . The growth factors EGF and basic fibroblast growth factor  were added at 10 ng/ml each. The monolayer cells were further passaged weekly until enough cells were generated. The cells were then frozen in liquid nitrogen and stored as banks of APCs. Thawed APCs were further expanded and allowed to differentiate into the committed AstroRx cells by the removal of growth factors (EGF and bFGF), 50 μg/ml ascorbic acid (Sigma) was added, and the culture was continued for 7 days to yield AstroRx cells.The protocol for manufacturing the human astrocytes (AstroRx®) from the embryonic stem cells was performed according to the protocol detailed in Izrael et al. . In brien = 10 mice per experimental arm). Naive mice  were used as a control. Treated animals received i.v. injection of AstroRx cells or PlasmaLyte 6 h after LPS-induction. All the animals were sacrificed 72 h after the LPS instillation. The measurements of survival, body weight, hematology, lung histopathology, flow cytometry analysis of immune cells as well as cytokine and chemokines were collected from the BALF and serum. BALF was collected by intratracheal injection of 0.5 ml phosphate buffered saline (PBS) with 0.1 mM EDTA followed by a gentle aspiration three times. The recovered fluid was pooled and centrifuged. The BALF supernatant was preserved for the measurement of cytokines and chemokines. The sediment cells were resuspended and subjected to flow cytometry analysis.Female, 8 weeks old, BALB/C mice were obtained from Envigo (Israel) and maintained in SIA facility . Animal handling was performed according to the guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The experiment was performed under the approval of “The Israel Board for Animal Experiments.” A total of 30 BALB/C female mice were anesthetized using isoflurane and treated through intratracheal route of administration with LPS  to induce ARDS, the animals were randomized to receive 200K, 500K AstroRx cells, or PlasmaLyte  at an objective magnification of × 4 and × 10 and microscope's Camera .An analysis for ALI was performed according to the method described in Matute-Bello et al. using a severity scoring scale of 0–2, based on the American Thoracic Society Documents, 2011 . An analNot visible within the field—a score of 0; 1–5 neutrophils per field−1; more than 5 neutrophils per field−2.Not visible within the field—a score of 0; a single well-formed band of fibrin within the airspace−1; multiple eosinophilic membranes−2.Due to technical artifacts, only septal thickening that is ≤ two times the normal was considered. Less than ×2—score 0; ×2– ×4—score 1; more than × 4—score 2.The analysis was based on the measurements of 20 fields, using an objective magnification of ×4 and ×10 .2.Neutrophil cell count was performed using MATLAB color-based, brightness-based, and morphological-based segmentation. The cells were counted from a rectangle of 88,892 μmThe BALF cytokine concentrations were measured using the ProcartaPlex Luminex platform . The measurements were performed two times (25 μl of each sample) with a custom multiplex panel detecting the following mouse cytokines: IFNγ, TNFα, RANTES, IL-6, IL-10, IL-1α, IL-1β, IP-10, MIP1α, and MCP-1/CCL2. The measurements were performed using the Luminex MAGPIX instrument, and results were analyzed with Xponent 4.2 software according to the instructions from the manufacturer.Three female C57/Bl6 mice at age of 6–8 weeks were euthanized by injection of a lethal dose of Pental . The lymph nodes were excised and drained to obtain lymph-node cells (LNCs). The LNCs from all the mice were pooled and then counted by a hemocytometer using trypan blue dye to exclude the dead cells. One million LNCs were seeded in RPMI-1640 medium  supplemented with 2.5% fetal calf serum , 1 mM L-glutamine , and penicillin-streptomycin . The LNC cultures under the multiple experimental conditions were maintained in flat-bottom plates, in duplicates, in a humidified atmosphere of 5% carbon dioxide at 37°C. The mNPCs were obtained using the method described in Fainstein and Ben-Hur  for isol+/CD25+) and proliferating T cells (CD3+/BrdU+) of the LNC cultures in the presence of ConA were set as baselines for the T-cell activation—without immunomodulation. The AstroRx® cells were added to LNC culture at ratios of 2:1, 5:1, and 10:1 LNC:AstroRx®, with and without 2.5 μg/ml ConA. Further, the LNCs in 0.5 ml RPMI-1640 were cultured with 0.5 ml conditioned medium with and without 2.5 μg/ml ConA. As a positive control, the mNPCs were added to the LNC culture at a ratio of 2:1 with and without 2.5 μg/ml ConA.Co-expression of CD3/BrdU determined the percentage of proliferating T cells in co-cultures with LNC. The LNCs were kept in culture with and without 2.5 μg/ml ConA  for 48 h. The percentage of T cells , anti-CD44 , anti-CXCR4 , anti-TRA-1-60 , anti-EPCAM , anti-SSEA4 , anti-GFAP , anti-Nestin , and anti-AQP-4 . The Flow Cytometer FACS Canto II  was operated with FACSDIVA software (BD). At least 10,000 events were collected per sample. For immune cells identity, the following antibodies were used: FITC Rat Anti-Mouse I-A/I-E Clone 2G9 (RUO) (BD 553623), PerCP-Cy™5.5 Hamster Anti-Mouse CD3e (BD 551163), PerCP-Cy™5.5 Rat Anti-Mouse CD45R/B220 (552771), Mouse CCR3 PE-conjugated Antibody (R&D FAB729P), and APC-anti-mouse CD11 (Biolegend BLG-117310d).p-values were calculated by Mann–Whitney test. The values ≤0.05 were considered significant .The statistical analyses were performed using GraphPad Prism 7 software . The We are currently evaluating the curative role of AstroRx in patients with ALS. In a Phase I/IIa, open-label, dose-escalating clinical study, we studied the safety, tolerability, and therapeutic effects of AstroRx intrathecal transplantation.Similarly to ARDS, the levels of circulating chemokines and cytokines in ALS are increased. The patients with a shorter diagnostic delay, which is a marker of more severe rapidly progressing disease  display In preclinical studies, the AstroRx cells demonstrated a neuron-protective phenotype (A2 astrocytes) and secreted neurotrophic factors that were capable to uptake glutamate and regulate oxidative stress . DependiTo test the immunomodulatory effect of AstroRx cells, we chose a rapid and acute inflammation model. LPS lung instillation is one of the most used models for ARDS. This model shares a number of pathological features with COVID-19-related ARDS, such as hypoxemia, neutrophil accumulation, alveolar space thickening, fibrin and tissue pathology, and high levels of inflammatory cytokines , 10, 13.via intravenous infusion allows wide distribution throughout the body. The cells administered intravenously would encounter the first-class pulmonary effect . Comprehensive safety investigation on the fate of AstroRx cells was done once the cells were injected intrathecally into the cerebrospinal fluid . The safThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The animal study was performed according to guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The experiment was performed under the approval of The Israel Board for Animal Experiments.MR, MI, AR, and JMW conceived and designed the studies. MI, JMW, SGS, TS, and KM performed the experiments, analyzed the data, and interpreted the data. MR, MI, and JMW wrote the manuscript. All authors read and approved the final manuscript.MI, AR, SGS, KM, TS, JMW, and MR are employed by company Kadimastem Ltd.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Validation of self-reported tools, such as physical activity (PA) questionnaires, is crucial. The aim of this study was to determine test-retest reliability, internal consistency, and the concurrent, construct, and predictive validity of the short semi-quantitative Physical Activity Unit 7 item Screener (PAU-7S), using accelerometry as the reference measurement. The effect of linear calibration on PAU-7S validity was tested.A randomized sample of 321 healthy children aged 8–16 years  from the nationwide representative PASOS study completed the PAU-7S before and after wearing an accelerometer for at least 7 consecutive days. Weight, height, and waist circumference were measured. Cronbach alpha was calculated for internal consistency. Test-retest reliability was determined by intra-class correlation (ICC). Concurrent validity was assessed by ICC and Spearman correlation coefficient between moderate to vigorous PA (MVPA) derived by the PAU-7S and by accelerometer. Concordance between both methods was analyzed by absolute agreement, weighted kappa, and Bland-Altman statistics. Multiple linear regression models were fitted for construct validity and predictive validity was determined by leave-one-out cross-validation.p < 0.001). Spearman correlation and ICC coefficients of MVPA derived by the PAU-7S and accelerometers increased from 0.31 to 0.62 and 0.20 to 0.62, respectively, after calibration of the PAU-7S. Between-methods concordance improved from a weighted kappa of 0.24 to 0.50 after calibration. A slight reduction in ICC, from 0.62 to 0.60, yielded good predictive validity. Multiple linear regression models showed an inverse association of MVPA with standardized body mass index  and waist to height ratio . All validity dimensions were somewhat stronger in boys compared to girls.The PAU-7S overestimated MVPA by 18%, compared to accelerometers . A Cronbach alpha of 0.76 showed an acceptable internal consistency of the PAU-7S. Test-retest reliability was good (ICC 0.71 The PAU-7S shows a good test-retest reliability and acceptable internal consistency. All dimensions of validity increased from poor/fair to moderate/good after calibration. The PAU-7S is a valid instrument for measuring MVPA in children and adolescents.Trial registration numberISRCTN34251612.The online version contains supplementary material available at 10.1186/s12966-021-01169-w. Physical activity (PA) is associated with favorable mental and physical health in children and adolescents –3. The W. Therefore, brief PA questionnaires are needed to readily identify children not meeting the WHO PA recommendations and assist in PA counseling [Measurement of daily PA is paramount to identify children not meeting current recommendations and implement intervention programs aimed to promote PA that can engage this at-risk population. However, the measurement of true PA is challenging. Objective methods to measure PA, such as accelerometry, are difficult to implement in large-scale epidemiological studies and time-limited settings due to economic and logistic burdens , 7. Addiunseling . For thiPA questionnaires are generally developed specifically for each study population and research aim due to the impact of ethnicity, culture, behavior, and biology on PA (7). Therefore, the validity of PA questionnaires beyond the target study population is limited. Furthermore, measurement error of self-reported data is a concern. The available calibrated questionnaire-derived PA data, although scant, show promising results , 14–18.The aim of the present study was to determine the reliability and the concurrent, construct, and predictive validity of PAU-7S in a randomized, nationally representative subsample of the PASOS study of children and adolescents aged 8 to 16 years. Additionally, we evaluated the effect of linear regression calibration on each of the three dimensions of PAU-7S validity.This validation study was performed within the frame of a nationwide representative study of Physical Activity, Sedentarism, lifestyles and Obesity in Spanish youth (PASOS). The methodology of the PASOS study has been described in detail elsewhere . In brie. The study protocol was approved by the Ethics Committee CEIm Fundació Sant Joan de Déu, Spain . Parental written informed consent was obtained.Of this study population, a randomized sample of 389 (10.2%) children and adolescents was invited to participate in the validation study and 369 (94.9% response rate) agreed to participate. For test-retest reliability analysis, 321 participants completed PAU-7S questionnaires at baseline and after 1 week of wearing an accelerometer. After excluding 17 participants with missing or invalid accelerometer data, 304 participants were included in the validity analysis. Participants did not receive any compensation for their participationThe development of the PAU-7S involved three strategies: (i) review of validated PA questionnaires for children; (ii) consulting PA experts from the IMIM-Hospital del Mar Research Institute; (iii) analysis of PA data of the Thao-POIBC  and EnKi.The resulting 7-day PA questionnaire was designed to measure regular PA in a typical week. Most PA questionnaires commonly used with children focus on this timeframe because they show good weekly recall . The PAUThe PAU-7S was administered the first day the accelerometer was worn and 9 days later, when the accelerometer was taken off by trained personnel. MVPA was calculated based on the sum of all activities, with the exception of walking.PA was measured by the “Actigraph GT3X+” accelerometer , allocating at least 7 days from April to June 2019 for each of the 22 randomized school groups. Children were asked to wear the accelerometer for at least 1 week except while bathing or swimming. The accelerometers were placed on the wrist for the non-dominant hand with a bracelet. The accelerometer data collection protocol was followed by all field workers. A common training session was carried out to ensure the homogeneity of this procedure. The Troiano et al. method  was usedwww.datahunter.es) was used to identify wear-time between 8 a.m. and 10 p.m.The Actigraph data were downloaded using the software provided by the manufacturer  and imported into SPSS v21  for data processing and screening. R package 4.0.2 accelerator  measurements were taken by trained personnel, with the children in light clothing, without shoes. The measurements were performed using an electronic SECA 899 scale (recorded to the nearest 100 g), a portable SECA 217 stadiometer (to the nearest 1 mm), and a flexible, non-stretch SECA 201 metric tape (to the nearest 1 mm), respectively. WC was measured in the narrowest zone between the lower costal rib and iliac crest, in the supine decubitus and horizontal positions. BMI z-score was computed using age and sex-specific reference values from the WHO . Waist tFollowing anthropometric and initial weight, height, and WC measurements, participants completed the first PAU-7S during a group session in the computer room at school (1st PAU-7S). Upon questionnaire completion, they received an accelerometer and verbal instructions on its use. Nine days later, participants again completed the questionnaire (2nd PAU-7S).Participant characteristics were described as mean, standard deviation (SD), and median (inter-quartile range), as appropriate. Distribution of continuous variables between boys and girls were compared using the Student t test for normally distributed variables or Mann-Whitney U test otherwise. Proportion comparisons for categorical variables were assessed using chi-square test. Non-normally distributed variables were log-transformed to achieve normality.Internal consistency of the PAU-7S questionnaire was tested by Cronbach alpha. Test-retest reliability between PAU-7S-derived basal and 1-week MVPA data was assessed by intra-class correlation coefficients (ICC).The relative validity of the PAU-7S was assessed by Pearson correlation coefficients comparing MVPA derived by the PAU-7S (test method) to that shown by the accelerometers . Pearson correlation coefficients were classified as follows: > 0.8, very good; 0.61–0.80, good; 0.41–0.60, moderate; 0.21–0.40, fair; and < =0.20, weak . AlthougWe further assessed agreement between the two measurements using the original Bland–Altman method  and a moEnergy balance is the ratio between energy intake and energy expenditure. Energy expenditure increases with PA , 30, whiTo reduce measurement error of the PAU-7S, we performed linear calibration according to Rosner et al. . MVPA deThe predictive capacity of the calibration equation was assessed by leave-one-out cross-validation. This iterative procedure predicts the response value of each individual from the model fitted by the rest of the sample. The classification system of interaction between the PAU-7S-derived MVPA, sex, and age was tested.p < 0.05.The Statistical Package for the Social Sciences statistical software package version 21.0  was used for all statistical analyses with the exception of leave-one-out cross-validation. This analysis was performed using R package 4.0.2. Differences were considered significant if n = 323) and those of the remaining PASOS cohort (n = 3496) revealed no significant differences with the exception of age. Participants in the validation study were somewhat younger (12.3 ± 2.2 years) than the remaining participants of the PASOS cohort (12.6 ± 2.4) (Supplementary Table ). No significant difference between the sample with accelerometer data (n = 304) and the sample for reliability analysis (n = 321) was found.Characteristics of the study population are reported in Table (Table ). The repeatability of each PA activity ranged from moderate to good.The PAU-7S showed good test-retest repeatability for total PA and MVPA in both boys and girls Table . The rep(Table ). The noncalibrated PAU-7S significantly overestimated MVPA (by 18%) compared to the criterion standard for validity; furthermore, this discrepancy significantly increased  with higher levels of MVPA (Table ). Overestimation of MVPA was somewhat greater in boys than in girls (Table ) and in adolescents compared to children (Supplementary Table ). Table . Additional kappa statistics, which account for agreement by chance, showed fair concordance between the two methods, with the identical kappa value in boys and girls (k = 0.24) (Table ).The absolute agreement of the PAU-7S as measured by correct cross-classification of tercile distribution of MVPA by the two methods was 46.7% for the entire population; somewhat higher values were observed for boys (48.2%) compared to girls (45.2%) Table . Additio4) Table .p = 0.014) and standardized BMI (zBMI) (p = 0.077), as shown in Table Multiple linear regression models adjusted for sex and age revealed an inverse association of MVPA derived by PAU-7S with WHtR p = 0.01 and stan(Table ). Pearson and ICC coefficients increased to 0.62 for both of these dimensions, with similar results in both boys and girls. The concordance between methods as measured by absolute agreement and kappa statistic improved to 59.2% and 0.50, respectively. The regression coefficients of the MVPA association with zBMI and WHtR slightly increased after calibration of the PAU-7S (Table ).Concurrent validity considerably improved after linear calibration of the PAU-7S Table . Pearson7S Table .p < 0–05) and 1.9 * 10− 6 (p > 0.05) min/d per each minute of the mean MVPA obtained by both methods, for the non-calibrated and calibrated PAU7s, respectively. This fact indicates that the calibration of the questionnaire eliminated the proportional bias.The mean agreement between MVPA reported by accelerometers  is shown in Figs. Cross-validation analysis of the predictive validity of the PAU-7S showed a slight reduction of Pearson and ICC coefficients from the original validation sample to the test sample from 0.62  to 0.60  and from 0.62  to 0.60 .The PAU-7S showed a good test-retest reliability and acceptable internal consistency. The questionnaire fairly ranked children according to levels of MVPA, with slightly better results in boys and adolescents compared to girls. Most importantly, although the noncalibrated PAU-7S overestimated MVPA, especially at higher levels of activity, linear calibration meaningfully increased the concurrent and construct validity of the questionnaire.In general, the ability of PA questionnaires to adequately measure PA in children and adolescents is modest at best , 33. AltThe consistency of participant responses across the items of the PAU-7S was determined by Cronbach alpha. In general, all the questionnaire items are supposed to reflect the same underlying construct, and therefore should be correlated with each others . The intIn the noncalibrated PAU-7S data, MVPA was overestimated by 11.4 min per day in comparison to accelerometer-derived MVPA. This is considerably lower than the IPAQ-A overestimation by 39.8 min of MVPA in Spanish adolescents . FurtherThe Spearman correlation of 0.31 for concurrent validity in the present study falls within the range reported for most PA questionnaires for youth , 33; we In most of the previously published studies, determination of the validity of the questionnaires used was limited to the assessment of concurrent validity by Spearman or Pearson correlation coefficients. This analysis can yield insights into the capacity of a questionnaire to rank children according to PA levels  but cannot assess absolute agreement between the PA questionnaire and the criterion standard of validity. In the present study, we found a somewhat lower ICC of MVPA between methods compared to that reported by Martín-Bello and colleagues  for the Relatively few studies have addressed this issue for self-reported PA assessment in children (11–16). In our study, Pearson and Spearman correlation coefficients increased to 0.63 and 0.62, respectively, after calibration of PAU-7S-derived MVPA estimates. Absolute agreement between methods was moderate (kappa = 0.50) for calibrated estimates of MVPA. Furthermore, the beta coefficient of the association between MVPA derived by the PAU-7S and the WHtR and zBMI score increased from − 0.010 to − 2.46 and from − 0.162 to − 5.850, respectively. This finding indicates a considerable improvement of the construct validity of the PAU-7S after calibration. Finally, the predictive validity of the calibrated PAU-7S data was good, according to cross-validation with an independent internal sample. These results clearly show that linear calibration of MVPA derived by the PAU-7S strongly improved all validity dimensions tested. This finding is in line with the scarce evidence from other PA questionnaire calibration studies in children . Saint-Maurice and colleagues found a reasonable ability of the PAQ-C and PAQ-A calibration algorithm to estimate group-level estimates of accelerometer activity (15). Similar results were reported for the YAP questionnaires (14), the Global Physical Activity Questionnaire (12), and the Previous Day Physical Activity Recall (11) calibration algorithm.The main strength of the present study is the population-based design, which permits generalization of the results to other Spanish populations of children aged 8–16 years. The inclusion of multiple dimensions of validity –concurrent, construct, and predictive– also can be considered a strength of this study. The short length of the PAU-7S makes it ideal for time-limited settings such as primary care centres and for large epidemiological studies or those that attempt to evaluate a long list of indicators. In addition, it will be useful for monitoring national PA data and for comparison with other countries. Finally, these results provide evidence that calibration can improve the validity of PA questionnaires for children and adolescents. This study also has two limitations that must be noted. Accelerometers are not sensitive to PA such as cycling or aquatics (7), and measurement error of self-reported data is an inherent limitation of questionnaires. Furthermore, the PAU-7S includes only 6 general questions about physical activities, which allows only a rough overview of the PA pattern and of METs spent in physical activities. For example, METs of sport activities are specific for each sport but the PAU-7S asks globally for time spent in team and individual sport activities. Therefore, the PAU-7S contribution to calculating the corresponding METs of these activities is limited. Hence, it should not be used when a more exact estimation of METs is the purpose of the study. Furthermore, a single administration of the PAU-7S will not accurately reveal seasonal variation in physical activities.The PAU-7S is a valid instrument for the measurement of physical activity in Spanish children and adolescents aged 8 to 16 years. The questionnaire is an adequate instrument for a general estimation of PA, especially in time-limited settings such as primary care and in epidemiological surveys with a large sample size or with many measures of other health indicators where the administration of accelerometers is not feasible. The calibration of this questionnaire meaningfully decreased measurement error and thereby increased its validity. Further studies are needed to shed light on the external validity of the PAU-7S.Additional file 1 Supplementary Table 1. Characteristics of the validation study participants and the remaining participants of the population-based PASOS cohort.Additional file 2 Supplementary Table 2. Correlation coefficients and between-method agreement of moderate to vigorous physical activity measurements derived by the Physical Activity Unit 7-item screener, noncalibrated and calibrated, and the reference method (accelerometer), stratified by age.


Cancer cachexia worsens the treatment outcomes of patients with small-cell lung cancer (SCLC). However, no reliable biomarker of cancer cachexia is yet known.We retrospectively evaluated male SCLC patients who received induction chemotherapy or concurrent chemoradiotherapy. The cachexia index (CXI) was calculated as skeletal muscle index × serum albumin level (g/dL)/neutrophil-to-lymphocyte ratio. The CXI cutoff according to tumor stage was determined based on a time-dependent receiver operating characteristic curve, and all patients were divided into low- and high-CXI groups.p = 0.004). More low-CXI patients required early discontinuation of treatment because of treatment-related toxicity compared to the high-CXI patients . The median progression-free survival (PFS) and overall survival (OS) were significantly shorter in the low-CXI group than the high-CXI group . On multivariate analysis, low-CXI status was an independent poor prognostic factor for both PFS and OS regardless of the tumor stage.Of 267 patients, 83 and 24 patients with limited-stage disease (LD) and 123 and 37 patients with extensive-stage disease (ED) were assigned to the high- and low-CXI groups, respectively. Only one of 24 patients (4.2%) with LD in the low-CXI group achieved a complete response (CR), whereas 30 of 83 patients (36.1%) with LD in the high-CXI group achieved CRs (A low CXI was associated with treatment intolerance, poor treatment response rate, and poor prognosis in SCLC.The online version contains supplementary material available at 10.1186/s12885-021-08300-x. Small cell lung cancer (SCLC) is a highly aggressive disease characterized by rapid tumor growth, early widespread dissemination, and a high probability of relapse , 2. AlthCancer cachexia  is assoFrom July 2006 to June 2020, all consecutive male SCLC patients receiving etoposide or irinotecan plus platinum combination chemotherapy as first-line treatment (with or without radiotherapy) in a single institution were retrospectively reviewed. As the CXI cutoff likely differs according to sex because muscle mass varies by sex, female patients were excluded as the sample size was too small to determine their cutoff. Patients with another type of cancer and/or a serious active infection were excluded. Those for whom serum albumin levels and complete blood counts measured within 7 days before the first cycle of chemotherapy as well as baseline chest computed tomography (CT) scans were unavailable were excluded. The study was approved by the Institutional Review Board of Gyeongsang National University Hospital.2); the SMI thus had the unit cm2/m2. Underweight was defined as a body mass index (BMI) <  18.5 kg/m2 (the Asian criterion). The response to anticancer therapy was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) ver. 1.1  for LD and 0.665 (95% CI 0.577–0.753) for ED  and median (interquartile range) CXI values were 11.09 (± 6.17) and 10.08 (6.30–14.24) for LD patients and 8.18 (± 5.92) and 6.77 (3.94–11.06) for ED patients, respectively (p = 0.033), whereas there was no difference between the two groups for ED patients. The proportion of patients classified as Eastern Cooperative Oncology Group (ECOG) PS 2–3 was higher in the low-CXI group than in the high-CXI group regardless of stage . Among ED patients, the low-CXI group received prophylactic cranial irradiation less frequently  and had a lower median BMI  compared to the high-CXI group.Comparison of patient baseline characteristics between the high- and low-CXI groups are presented in Table p = 0.026) for LD patients and 54.1 and 85.4% (p <  0.001) for ED patients, respectively. Only one LD patient and no ED patient achieved a CR in the low-CXI group. All 33 patients who achieved a CR completed their planned treatments. When 51 patients who discontinued treatment early (because of toxicity or patient decision) or for whom the radiological treatment response was not assessed were excluded from the analysis, the ORR remained lower in the low-CXI group compared to the high-CXI group regardless of stage .Of 267 patients, 16 were not available for the radiological assessment of treatment response  and discontinued treatment early because of treatment-related toxicity more frequently  compared to high-CXI patients. TRM tended to occur more frequently in the low-CXI group than in the high-CXI group for ED patients .Adverse treatment-related events are listed in Table p <  0.001; Fig. p <  0.001; Fig. p <  0.001; Fig. p <  0.001; Fig. The median follow-up duration was 41 months. Similar findings were obtained regardless of stage. In LD, patients with a low CXI had a shorter PFS  in LD patients. In ED patients, an ECOG PS of 2–3 and a low CXI were independent poor prognostic factors for PFS  and OS   2.445, 95% CI 1.406–4.253, This is the first study to report that a low CXI is closely related to poor clinical outcomes in SCLC patients. The CR rate and ORR differed greatly between the low- and high-CXI groups. Although the ECOG PS, which is an important prognostic factor, was not balanced between the two groups, PFS and OS were much poorer in the low-CXI group even after adjusting for the PS. Although the role for the CXI has not yet been examined in SCLC patients, each CXI factor has been suggested to be prognostic in several studies. Sarcopenic SCLC patients experienced poorer survival than non-sarcopenic patients . When moCancer cachexia must be carefully assessed. Percentage weight loss alone is of limited utility in the era of obesity , 37. SarThe low-CXI group very frequently discontinued treatment early because of treatment-related toxicity and suffered a higher rate of TRM. A recent study reported that malnourished patients exhibited increased rates of toxicity of grade ≥ 3 and were more likely to be hospitalized in phase I and II oncology clinical trials . SeveralOur work had several limitations. First, the data were collected retrospectively and thus associated with a risk of selection bias. We also lacked detailed toxicity profiles. Second, the utility of the CXI in female patients could not be assessed in this study. Third, the method used to measure the SMI in the present study was different from the methods used in previous studies, and calculation of the CXI using the SMI of pectoralis muscle was not validated in SCLC, rendering comparisons difficult. Given the low sensitivity of the CXI cutoffs in the present study, validation of our methodological approach is essential. In the original study, Jafri et al. calculated the CXI and SMI using the skeletal muscle area as determined on abdominal CT in non-small cell lung cancer patients . In our In conclusion, we found that cachectic patients with low CXIs more frequently discontinued treatment early and exhibited poor prognosis in SCLC. Intensive supportive care, including aggressive nutritional support and dose adjustment, may improve the treatment outcomes of SCLC patients with a low CXI receiving chemotherapy or chemoradiotherapy. We anticipate that the CXI may be adopted as an ideal biomarker for research on cancer cachexia and that its measurement will be integrated into routine practice for the diagnostic work-up for SCLC after validation through further prospective studies.Additional file 1. Time-dependent receiver operating characteristic (ROC) curve of the cachexia index (CXI) for prediction of (A) 18-month overall survival in limited-stage disease (LD) and (B) 10-month overall survival in extensive-stage disease (ED). Circles indicate CXIs of (A) 5.82 in LD and (B) 3.83 in ED. These cutoffs were determined by maximizing the Youden index.


MicroRNAs (miRs) are a group of endogenous, non-coding, 18–24 nucleotide length single-strand RNAs. These molecules mediate the gene expression and are involved in regulating diverse cellular biological processes, i.e. cell cycle, differentiation, and apoptosis. Aberrant  miR-138 and miR-424-5p expression by using real-time PCR and ΔΔCT. Blood samples were obtained from 30 patients (15 cases and 15 controls), to determinemiR-424-5p expression was 1.96 times higher in the case group, but miR-138 expression was 3.05 times lower in the plasma of OSCC patients. The median CT values of miR-138 were 27.60 and 28.70, while those of miR 424-5p were 29.40 and 30.0 in the case and control groups, respectively. Mann-Whitney test indicated no significant difference in miR-138 and miR-424-5p between the two groups (P > 0.05). However, results obtained by ΔΔCT method showed that miR-138 and miR-424-5p expression in serum can be used as potent markers for carcinoma detection and also may be a potentially therapeutic approach in the future. Further longitudinal studies with larger samples are required to verify these findings.Our findings suggest that the evaluation of  Oral squamous cell carcinoma (OSCC) is one of the life-threatening cancer types worldwide, with a high frequency of recurrence and a low survival rate . Notwithstanding many efforts made to improve the methods for treating OSCC, the therapeutic efficacy and the five-year survival rate remain unsatisfactory . Therefore, more understanding of the molecular pathologies underlying OSCC provides a novel insight into the development of effective therapeutic strategies for OSCC.  miR expression has been suggested to be an important event in the pathologies of various types of cancer, particularly OSCC .In recent years, microRNAs (miRs), a small type of non-coding RNAs, have been found to play a significant role in many diseases, including cancer . MiRs are a group of endogenous, non-coding, 18–24 nucleotide length single-strand RNAs that mediate the gene expression. These RNAs are involved in regulating diverse cellular biological processes, including cell cycle, differentiation, and apoptosis. AberrantmiRs in OSCC represent new directions in the search of oral carcinogenesis . Although there are many studies on miRs in serum, plasma, and urine in different types of carcinoma such as OSCC, circulating miRs may play a significant role as diagnostic or prognostic biomarkers in human carcinomas . These magic molecules can also be responsible for oncogenes in the progression of cancer processes or tumor suppressor genes . The expression patterns of miR-424-5p significantly induces OSCC cell invasion and migration, demonstrating that miR-424-5p may function as an oncogenic miR and may contribute to the progression of OSCC. Furthermore, the IL-8-induced tumor cell migration and invasion were prevented when miR-424-5p was specifically inhibited by an antagomir (AM424), which indicates that the induction of miR-424-5p is essential for IL-8-induced cellular invasiveness . SOCS2, suppressor of the cytokine signaling, has been identified as a novel target of miR-424-5p, which its overexpression in oral cancer cells can promote the cell migration and invasion through SOCS2 repression .Emerging studies have shown that miR-138 acts as a tumor suppressor by targeting many genes through proliferation, apoptosis, invasion, and migration and also can sensitize tumors to chemotherapies . Findings have indicated that miR-138 has ability to inhibit OSCC cell movement and is able to significantly be downregulated in cancer tissues, but not adjacent normal tissues, in OSCC patients . Overexpression of miR-138 and miR-424-5p expression in OSCC patients in comparison with healthy control individuals, to identify whether these biomarkers could be diagnostic tools for the effective OSCC detection. Moreover, we investigated the difference in the expression of two miRs in patients with OSCC compared with healthy people, particularly in the early stage.In the present study, we analyzed circulating serum Sample collectionThis study included 15 patients with OSCC and 15 healthy controls . The bloSerum samples storage and total RNA extractionPeripheral blood samples were obtained before any therapeutic intervention, including surgery or preoperative radiation. All the samples were collected in clot stimulator tube. The basic clinical information was acquired from each participant. The blood was kept for 45 minutes to allow clotting for serum separation and then processed in accordance with the instruction recommended by Mircury Exiqon (Denmark). Thereafter, the blood samples were centrifuged at 2,500 ×g at 4°C for 15 minutes. Subsequently, the fluid was recentrifuged at 4°C at full spin for 15 minutes to remove the remnant contaminants, including erythrocytes. The serum was stored at -70°C until processing for total RNA isolation. As per the manufacturer’s protocol, total RNA was extracted from serum samples using Mircury Exiqon. For this purpose, the miRNeasy Mini Kits and miRNeasy serum (both from Mircury Exiqon) were employed to extract miRs from serum samples. According to the guidelines of manufacture, optical densities for RNA extraction were 260/280 ratio.cDNA synthesisAll the extracted cDNA were normalized to 2 μg, and then the cDNA synthesis was carried out by using cDNA Syntheses Kit . Synthesized cDNAs from the total RNA stem-loop were used in real-time RT-PCR. Reverse transcriptase (RT) reactions were comprised of the following reagents: 2 μg of RNA sample, 50 nmol/L of stem-loop RT primer, 2× RT buffer, 0.5 mmol/L of each dNTP, and 4 U/μL of M-MLV RT. Reactions (20 μL) were incubated in a PCR system at 37°C for 50 minutes and 85°C for 5 minutes. The samples were then held at 4°C.Real-time PCRReal-time PCR was performed using SYBR Green PCR Master Mix  on a real-time PCR instrument, consistent with manufacturer’s instruction. Each reaction was accomplished in a volume of 20 μL, containing 50 ng of cDNA, 2 μL of universal primer, and 10 pmol of each primer, along with 10 μL of 2× QuantiTect SYBR Green PCR Master Mix. The PCR amplification reaction consisted of denaturation at 95°C for 5 seconds, followed by 40 cycles at 62°C for 20 seconds and 72°C for 30 seconds. All reactions were carried out in triplicate. The comparative Ct method was applied to analyze the differences in each group in expression levels. Statistical analysisAll the statistical analyses were conducted by the aid of SPSS 21.0 Software. To compare the findings of the serum expression level of two miRs, Mann-Whitney test was utilized, and differences with a P-value < 0.05 were considered as statistically significant. Gene Runner software was also employed to design primers. Owing to applying Rest Software, the negative mark denotes the elevated gene expression. In total, 30 participants, including 15 OSCC patients and 15 healthy individuals, were enrolled in this study. The mean ages of the control and case groups were 53.67 ± 9.15 and 55.80 ± 11.19, respectively. In both groups, eight males (53.3%) and seven females (46.7%) participated.Real-time RT-PCR results miR-138 marker was detected in 10 out of 15 OSCC and in 12 out of 15 healthy samples, whereas that of miR-424-5p marker was identified in 13 out of 15 OSCC and in 9 out of 15 healthy samples. Statistical comparison showed a significant difference between the two groups of both miR-138 and miR-424-5p expression, using the Two-sample binomial test . The expression ofExpression level differences of markersmiR-424-5p expression was 1.96 times higher in the case group than the healthy one, and miR-138 expression was 3.05 times lower in the plasma of OSCC patients .miR-138 has an inhibitory role in OSCC cell movement, and its expression is significantly downregulated in cancer tissues, but not in adjacent normal tissues in patients with OSCC . In Liu et al.’s study, a panel of differentially expressed miRs was identified, i.e. the reduction of miR-138 in highly metastatic cells in head and neck squamous cell carcinoma (HNSCC) cell lines. Ectopic transfection of miR-138 suppressed the cell invasion and led to cell cycle arrest and apoptosis. MiR-138 reduction enhanced cell invasion and suppressed apoptosis. The same authors also suggested that miR-138 acts as a tumor suppresser and could be used as a therapeutic target for HNSCC patients at risk of metastasis . Yu et al. showed the downregulation of miR-138 in tongue SCC (TSCC) and concluded that miR-138 is responsible for TSCC cell migration and invasion by concurrently targeting Rho-related GTP-binding protein C and ROCK2. These researchers also deduced that miR-138 could be used as a novel therapeutic target for TSCC patients at risk of metastatic disease . Peng et al. have emphasized that the overexpression of miR-424-5p significantly induces OSCC cell invasion and migration, implying that miR-424-5p may act as an oncogenic miR and may contribute to the progression of OSCC . According to a meta-analysis result, miR 424-5p expression was upregulated in cancerous compared to non-cancerous tissue . In this study, we demonstrated that serum miR-138 and miR-424-5p could be considered as exceptional tumor biomarkers. Based on the results obtained from ΔΔCT method, miR-138 expression was downregulated in patients with OSCC (3.05 times), while miR-424-5p indicated upregulation in these patients (1.96 times), which supports previous reported results.MiRs with a length of 18-24 nucleotides are a series of endogenous non-coding RNAs. Their primary function is the regulation of gene expression and RNA silencing in post-transcriptional level . Previous surveys have reported that the deregulation of miRs is one of the main factors in the initiation and progression of varied cancer types . In addition, circulating miRs is stably preserved and measurable in blood and thus can serve as reliable biomarkers for the diagnosis or prognosis of a number of carcinomas , including OSCC , and also can suggest a field-effect in oral cancer . Zhang et al. have demonstrated that Considering that the overall survival of patients with OSCC is highly poor, screening novel diagnostic biomarkers seems to be an urgent need in confronting with OSCC patients. Blood, in comparison with other body fluids, is generally more applicable as it is readily attained in a non-invasive approachable manner and can be stored for a long time. Thus, since 2008, circulating miRs is known to be diagnostic biomarkers and important approaches for the blood-based detection of human cancer .The measurement of miRs in serum can assist in the earlier detection of cancers, including OSCC. Circulating miRs might serve as biomarkers for risk of OSCC development and prognosis, as well as response to treatment. Despite recent advances in the field of miRs, therapeutic utility of miRs requires further studies with relying on an precise insight into the real mechanisms of these RNAs in OSCC.miR-138 and miR-424-5p expression in serum can be used as potent markers for carcinoma detection and also may be a potentially therapeutic approach in the future. More longitudinal studies with larger samples are necessary to confirm these findings.In conclusion, the findings of the present study uncovered that the evaluation of The authors confirm contribution to the paper as follows: study conception and design: F.B, N.B; data collection: M.H, A.GH; analysis and interpretation of results: A.F, F.B, A.M; draft manuscript preparation: N.B, A.M. All authors reviewed the results and approved the final version of the manuscript.


The present study aimed to synthesize and equate the mechanical properties and dentin interaction of two adhesives; experimental adhesive (EA) and 5 wt.% reduced graphene oxide rGO) containing adhesive. Scanning electron microscopy (SEM)-Energy-dispersive X-ray spectroscopy (EDX), Micro-Raman spectroscopy, push-out bond strength test, and Fourier Transform Infrared (FTIR) spectroscopy were employed to study nano-bond strength, degree of conversion (DC), and adhesive-dentin interaction. The EA was prepared, and rGO particles were added to produce two adhesive groups, EA-rGO-0% (control) and rGO-5%. The canals of sixty roots were shaped and prepared, and fiber posts were cemented. The specimens were further alienated into groups based on the root canal disinfection technique, including 2.5% sodium hypochlorite (NaOCl), Photodynamic therapy (PDT), and ER-CR-YSGG laser (ECYL). The rGO nanoparticles were flake-shaped, and EDX confirmed the presence of carbon (C). Micro-Raman spectroscopy revealed distinct peaks for graphene. Push-out bond strength test demonstrated highest values for the EA-rGO-0% group after NaOCl and PDT conditioning whereas, rGO-5% showed higher values after ECYL conditioning. EA-rGO-0% presented greater DC than rGO-5% adhesive. The rGO-5% adhesive demonstrated comparable push-out bond strength and rheological properties to the controls. The rGO-5% demonstrated acceptable DC , appropriate dentin interaction, and resin tag establishment.  Dental caries is a multifactorial infectious disease that results in tooth structure loss . To restsp2 structures and physical properties are still recoverable, and it is also purer and contains more graphene properties than GO [Graphene-based materials (GBMs) have recently caught the attention of dental researchers. The GBMs are thermally and chemically stable and enjoy a high surface area . Among t than GO . Studies than GO ,23. Considering the beneficial properties of rGO, it can be postulated that their inclusion could improve dentin adhesives’ mechanical and rheological properties. Henceforth, the present study was aimed at developing an experimental adhesive (EA) containing rGO nanoparticles. It was hypothesized that the inclusion of rGO nanoparticles would improve dentin interaction, durability, bond strength, and rheological properties of the adhesive. Our study was directed at synthesizing and characterizing an EA containing rGO nanoparticles utilizing techniques like scanning electron microscopy (SEM)—Energy Dispersive X-ray (EDX) spectroscopy, Micro-Raman spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, and degree of conversion (DC) analysis. Ethical approval was acquired before the commencement of the study, and all ethical protocols were firmly followed. The teeth used in the current study were collected from the institute’s orthodontic clinics. The EA was synthesized following the prior recommendations of Ye et al. , and thew/v %) for the EA group, as suggested formerly by AlFawaz et al. [The rGO nanoparticles were commercially acquired  and then added to the adhesive. The rGO powder was incorporated in 2 mL ethanol inside a microvial and sonicated at 37 °C for 10 min in an ultrasonicator . rGO powder was mixed with the ethanol solvent of the formulated EA at 0% to produce EA-rGO-0% (control) and at 5 wt.% to yield rGO-5%. To ensure homogenous dispersion of the filler particles, the rGO nanoparticles were mixed in resin and sonicated for 10 min inside an ultrasonic bath . After sonication, this blend was placed inside an ultrasonic homogenizer (Q500 Sonica) to yield a homogenized mixture at a pulse on/off for one minute at room temperature. To certify that the rGO nanoparticles were evenly dispersed post-storing, the mix was re-homogenized in the same ultrasonic homogenizer after every single use. To protect the notion that precise quantity of rGO nanoparticles were incorporated in the EA, these particles were first weighed up (in milligrams), and their volume was then computed (in milliliters). The subsequent formula was used to calculate weight/volume %  were stored at 4 °C and used in our experiments within 14 days of their preparation.−2 for 20 s, polymerized from a distance of 10 mm. Before the SEM, the samples were placed on aluminum stubs and then sputtered with a gold layer for 2 min . The investigation was accomplished at an accelerating voltage of 30 kV utilizing SEM . A number of magnifications (selected on the basis of convenience) were utilized to take SEM micrographs of the specimens. To warrant the presence of rGO nanoparticles inside the adhesive, we used SEM-EDX spectroscopy. EDX spectroscopy also helped the current study in exploring the elemental distribution inside the adhesive. Micro-Raman spectroscopy was also executed to analyze rGO nanoparticles. A micro-Raman spectrophotometer  with software (Raman reader®) was employed to acquire Raman spectra(s). With the help of a 0.9 objective lens, the laser beam was secured, and 600 mW power was used. One minute scan was completed three times for rGO nanoparticles. The particulars of the spectra were attained with a laser beam wavelength of 532 nm, between 1000 and 1700 cm−1, with noise filtration.The characterization of rGO nanoparticles was achieved via multiple investigative techniques. To characterize morphology of the rGO nanoparticles, SEM technique was used. From both adhesive groups (EA-rGO-0% and rGO-5%), five specimens were synthesized and photo-polymerized with a dental curing light source  with an output of 600 mW·cm®, Waterlase I-Plus, MZ8, California, CA, USA), which formed the basis of our six study groups.Sixty single-rooted maxillary second premolars were chosen for our study after carefully examining their anatomical features. The standardization was achieved by excluding the teeth with curved roots and including the teeth with approximately similar root lengths (21 mm ± 1 mm). These teeth were disinfected for 2 days using 0.5% chloramine solution at 4 °C. Post-disinfection, the teeth were cleaned with distilled water for 5 min. The teeth were stored in 1% thymol solution before their use and were utilized within a month after their collection. The anatomical crowns of all the teeth were cut at the cement-enamel junction with a diamond saw  to acquire roots with a uniform length of 19 mm. The canals of the attained roots were shaped and prepared using a step-back technique 1 mm short of the radiographic apex. The cleaning and shaping of the canals were achieved via K-files  and Gates Glidden drills (MANI) Nos. 2, 3, and 4, correspondingly. Rinsing was achieved with 10 mL of 2.5% sodium hypochlorite (NaOCl) with a 10 mL BD disposable syringe. File No. 35 was designated as the master apical file. The canals were dried using paper points . To obturate the canals, gutta-percha (GapaDent) and AH26 sealer  were combined, and the lateral compaction method was used. After 24 h of the obturation, 10 mm of each canal length were prepared using peeso reamers (MANI) of Nos. 4, 3, and 2. Distinctive drills were given by the manufacturer of No. 100 fiber posts  to standardize and manage the procedures better. The specimens were then fixed in a condensation silicone mold , entailing a putty consistency. We utilized 70% ethanol and compressed air to clean and dry the posts, respectively. The specimens were randomly allocated based on the type of adhesive used (EA-rGO-0% or rGO-5%), resulting in 30 samples being assigned to each group. The samples were further separated into groups based on the root canal disinfection technique, including 2.5% NaOCl (control), Photodynamic therapy (PDT) with methylene blue photosensitizer (MBP), and ER-CR-YSGG laser (ECYL) (BiolaseGroup 1 (EA-NaOCl): The root canal dentin was irrigated with the help of 10 mL of 2.5% NaOCl using a 10-mL BD disposable syringe. Root dentin was treated with EA-rGO-0% adhesive before the cementation of the fiber post. Group 2 (EA-PDT): MBP photosensitizer consisting of 2% aqueous solution was utilized to plug the root canals for 5 min. Light source  with the power output of 150 mW and wavelength of 638 nm was used to introduce into the canals. The diode laser was used for 2.5 min of irradiation, stopped for 2.5 min, and then irradiation was achieved again for 2.5 min. This protocol was followed to stimulate free radical formation without immensely elevating the destructive temperature. The light was applied as such that it gave 2.5 min of irradiation followed by 2.5 min of stop. To provide a full 360° even radiation, an optical fiber  was used. The parameters of the laser encompassed a diameter of 200 mm and taper of 0.03 mm. The root dentin was treated with EA-rGO-0% adhesive before the cementation of the fiber post. Group 3 (EA-ECYL): The ECYL  with a frequency of 30 Hz, power of 0.5 W with water/airflow of 25%, with a tip diameter of 275 μm was used. The laser optical fiber was inserted into the full root canal, and irradiation was carried out from apical to the cervical axis in a spiral way with a 2 mm/sec ratio, and irrigation was performed using 2.5% NaOCl. Five cycles of irradiation were accomplished with a time interval of 20 sec in between each cycle, and the roots were cooled off between the cycles. As with the previous two groups, the root dentin was treated with EA-rGO-0% before the cementation of the fiber post. Group 4 (rGO-5%-NaOCl): Irrigation of the root dentin was carried out with 10 mL of 2.5% NaOCl using a 10-mL BD disposable syringe. rGO-5% adhesive was smeared to the root canal dentin preceding fiber post cementation. Group 5 (rGO-5%-PDT): Root canal dentin was treated with PDT as explained in the former section. rGO-5% adhesive was smeared to the root canal dentin before fiber post cementation. Group 6 (rGO-5%-ECYL): Root canal dentin was treated with ECYL as described before. rGO-5% adhesive was smeared to the root canal dentin earlier to fiber post cementation.Group 1 (EA-NaOCl): The root canal dentin was irrigated with the help of 10 mL of 2.5% NaOCl using a 10-mL BD disposable syringe. Root dentin was treated with EA-rGO-0% adhesive before the cementation of the fiber post. Group 2 (EA-PDT): MBP photosensitizer consisting of 2% aqueous solution was utilized to plug the root canals for 5 min. Light source  with the power output of 150 mW and wavelength of 638 nm was used to introduce into the canals. The diode laser was used for 2.5 min of irradiation, stopped for 2.5 min, and then irradiation was achieved again for 2.5 min. This protocol was followed to stimulate free radical formation without immensely elevating the destructive temperature. The light was applied as such that it gave 2.5 min of irradiation followed by 2.5 min of stop. To provide a full 360° even radiation, an optical fiber  was used. The parameters of the laser encompassed a diameter of 200 mm and taper of 0.03 mm. The root dentin was treated with EA-rGO-0% adhesive before the cementation of the fiber post. Group 3 (EA-ECYL): The ECYL  with a frequency of 30 Hz, power of 0.5 W with water/airflow of 25%, with a tip diameter of 275 μm was used. The laser optical fiber was inserted into the full root canal, and irradiation was carried out from apical to the cervical axis in a spiral way with a 2 mm/sec ratio, and irrigation was performed using 2.5% NaOCl. Five cycles of irradiation were accomplished with a time interval of 20 sec in between each cycle, and the roots were cooled off between the cycles. As with the previous two groups, the root dentin was treated with EA-rGO-0% before the cementation of the fiber post. Group 4 (rGO-5%-NaOCl): Irrigation of the root dentin was carried out with 10 mL of 2.5% NaOCl using a 10-mL BD disposable syringe. rGO-5% adhesive was smeared to the root canal dentin preceding fiber post cementation. Group 5 (rGO-5%-PDT): Root canal dentin was treated with PDT as explained in the former section. rGO-5% adhesive was smeared to the root canal dentin before fiber post cementation. Group 6 (rGO-5%-ECYL): Root canal dentin was treated with ECYL as described before. rGO-5% adhesive was smeared to the root canal dentin earlier to fiber post cementation.2) signify the maximum failure load and post segments bonding area, correspondingly. To calculate the thickness of the slices and post segments diameter , digital calipers were employed, and the formula used for estimating the bonding surface wash = slice thickness.The roots containing cemented posts were sectioned vertically with the help of a Low-speed diamond saw  operated under water-cooling. This segmenting helped to achieve 0.5 mm thick six serial perpendicular slices from each root sample . Push-out bond strength was evaluated with the help of a universal testing machine . Slices gained through cutting were separately attached to the machine. The apical surface of the slices was subjected to loading while facing a cylindrical plunger with a metal rod  until failure was noticed. The crosshead speed used for this step was 0.5 mm/min. In total, 24 slices  were exposed to push-out testing. The formula used to estimate push-out bond strength wasThe nature of the bond failure was also investigated in our study for the cervical and apical root dentin with the help of a digital microscope . The failure modes were classified into adhesive-post, adhesive-dentin, and mixed types.SEM was used to assess the root dentin interface of ten segmented root dentin samples from each group. The samples were fixed on aluminum stubs and sputtered with a gold layer and witnessed under SEM . The accelerating voltage of the SEM was 30 kV whereas, different magnifications (based on convenience) were utilized.−1) and C = C  absorbance peaks (1638 cm−1) were acquired. The transformation rates for the adhesives were estimated using the ratios of C=C and C-C absorbance strengths (% of unreacted double bonds) pre-and post-curing by means of the following equation, recommended earlier by Al-Hamdan et al. [−1 absorption peak after curing, C aromatic is 1607 cm−1 absorption peak after curing, U-aliphatic is 1638 cm−1 absorption peak before curing and U-aromatic is 1607 cm−1 absorption peak before curing.The specimen size was 10 × 2 × 2 mm. FTIR spectroscopy was employed to estimate the DC of both the adhesive groups (EA-rGO-0% and rGO-5%). The steps of the FTIR spectroscopy mentioned in our previous study  were pren et al. :DC = [1 Utilizing an MCR-72 rheometer , the rheological properties of the composites were examined at rotation mode in a frequency sweep predetermined state of 8 mm parallel plate, 0.25 mm opening, and over an array of angular frequencies stretching between 0.1–100 rad/s at 25 °C.The statistical analysis was carried out for the push-out bond strength and DC analysis values utilizing SPSS-20.0 . Mean and standard deviation values were calculated, and the ANOVA and post hoc multiple comparison tests were utilized. The level of significance was set at 1%.−1 and 1584 cm−1, correspondingly. Characteristically, the D band represents sp3-hybridized carbon and deficits associated with grain boundaries and vacancies in rGO, while the G band signifies the vibration of sp2-hybridized carbon.The representative SEM micrograph demonstrated flake-shaped rGO nanoparticles with coarse ends, which were 1–2 nm in size . The EDXThe representative SEM micrographs illustrating resin tag formation for rGO-5% adhesive are presented in p > 0.01). The inter-group comparisons were statistically significant (p < 0.01) when EA-rGO-0%-NaOCl and EA-rGO-0%-PDT values were compared with EA-rGO-0%-ECYL values. The inter-group comparisons were also statistically significant (p < 0.01) when rGO-5%-NaOCl and rGO-5%-PDT values were compared with rGO-5%-ECYL values.The outcomes of the push-out bond strength (presented as means ± standard deviations) are presented in The percentages of interfacial failure types observed among different study groups of our study are presented in −1 and 1638 cm−1 wavenumbers. EA-rGO-0% presented with a higher DC (57.8 ± 8.7) as compared to rGO-5% (51.3 ± 6.6). No statistically significant results (p < 0.01) were perceived when the DC values of these two groups were compared (The characteristic FTIR spectra after the mixing of rGO nanoparticles (for cured and uncured adhesive) are shown in compared .The complex viscosities versus angular frequency of the two adhesive groups (EA-rGO-0% and rGO-5%) at 0.001 to 1000 rads/s are shown in Based on our study results, we can partially accept the hypothesis that the addition of rGO-5% nanoparticles improved the adhesive’s dentin interaction and bond strength. The hypothesis is also partially rejected as the DC of the adhesive decreased, and lower complex viscosity of the rGO-5% group were observed when matched with the EA-rGO-0% group. The clinical success of a restoration is particularly reliant on the adhesive properties, and a compromised adhesive-dentin bond could cause micro-leakage, development of secondary caries, and eventually, failure of the restoration . Previou−1 whereas the G band was perceived at 1584 cm−1, and this particular finding was similar to a previous study [The rGO nanoparticles, when observed on the SEM, revealed flake-shaped morphology, and this finding is consistent with several previous studies ,32. In gus study . For theus study . Concernus study . Anotherus study . These rus study .Push out bond strength is a consistent and practical method to test the adaptation strength of a material to its neighboring root dentin . Our pusConcerning interfacial failure types, most of the failures witnessed in our study were of the adhesive-dentin type, followed by mixed, and a few were adhesive-post type. These findings conformed with several previous studies ,45. AdheDC of the two adhesive groups was also evaluated in our study, and although the results demonstrated comparable DC for both adhesive groups, still, higher DC values for EA-rGO-0% adhesive were seen compared with rGO-5% adhesive. Earlier, Bin-Shuwaish et al. also reported an inverse positive linear relationship between increasing GO content and DC , and ourIn relation to the rheological properties, two adhesive groups demonstrated reduced viscosity as the frequency increased, and rGO-5% demonstrated lower complex viscosity values were when matched with EA-rGO-0% at various equivalent angular frequencies. Comparable findings were previously conveyed by Alrahlah et al. who revealed that the decrease in viscosity could be linked to increasing rGO filler content . This reThe findings of the current study, although encouraging, should be cautiously interpreted. The rGO-5% adhesive demonstrated comparable push-out bond strength and resin tag formation to the control group; however, a decreased DC was observed. As poor DC influences durability and water sorption of adhesive, the properties of adhesives with variable aging techniques should be explored in future studies. Moreover, studies aimed at exploring the effect of lower concentration of rGO nanoparticles in the adhesive should be carried out to further investigate this area.The rGO-5% adhesive presented comparable push-out bond strength and dentin interaction that matched the control group. However, a lower DC was detected for rGO-5% adhesive. The push-out bond strength was highest after NaOCl conditioning, followed by PDT and ECYL treatment for both adhesive groups. rGO could prove to be a promising filler in dentin adhesives, and future work is encouraged to probe the effect of various concentrations of rGO nanoparticles on the adhesive’s properties.